title: Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.
abstract: In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)-RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase-conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Sandra G
authors.initials: SG
authors.lastname: Zimmerman
authors.forename: Nathaniel C
authors.initials: NC
authors.lastname: Peters
authors.forename: Ariel E
authors.initials: AE
authors.lastname: Altaras
authors.forename: Celeste A
authors.initials: CA
authors.lastname: Berg
mesh_terms: D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D005260:Female; D017403:In Situ Hybridization / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D010053:Ovary / Q000187:drug effects / Q000378:metabolism*; D012313:RNA / Q000032:analysis / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014992:Xylenes / Q000494:pharmacology
chemical_list: D003902:Detergents; D016601:RNA-Binding Proteins; D014992:Xylenes; D012313:RNA